QIAGEN Plasmid Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Transfection efficiency versus plasmid purification method.

Different pRSVcat DNA preparations using the methods indicated were introduced into the indicated cell lines by liposome-mediated transfection, and the efficiencies determined by measuring CAT expression levels after 40 h. Each bar represents the mean of 4 independent transfections (2 transfections with each of 2 independent plasmid preparations). The highest transfection efficiency was achieved with QIAGEN Plasmid Kits.

QIAGEN Plasmid Kit procedures.

Neutralized bacterial lysates are cleared by centrifugation. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

The unique anion-exchange resin in QIAGEN-tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Prepacked QIAGEN-tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.

Specifications

Features

Plasmid
Giga Kit

Plasmid
Mega Kit

Plasmid
Maxi Kit

Plasmid
Midi Kit

Plasmid
Mini Kit

Applications

Transfection, cloning, sequencing, capillary sequencing, etc.

Transfection, cloning, sequencing, capillary sequencing, etc.

Transfection, cloning, sequencing, capillary sequencing, etc.

Transfection, cloning, sequencing, capillary sequencing, etc.

Transfection, cloning, sequencing, capillary sequencing, etc.

Culture volume/starting material

2.5–5 liters culture volume

500 ml – 2.5 liters culture volume

100–500 ml culture volume

25–100 ml culture volume

3–10 ml culture volume

Elution volume

Variable

Variable

Variable

Variable

Variable

Plasmid type

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

High-copy, low-copy, cosmid DNA

Processing

Manual (centrifugation)

Manual (centrifugation)

Manual (centrifugation)

Manual (centrifugation)

Manual (centrifugation)

Sample per run

1 sample per run

1 sample per run

1 sample per run

1 sample per run

1 sample per run

Technology

Anion-exchange technology

Anion-exchange technology

Anion-exchange technology

Anion-exchange technology

Anion-exchange technology

Time per run

320 min

220 min

160 min

150 min

80 min

Yield

<10 mg

<2.5 mg

<500 µg

up to 100 µg

<20 µg

Procedure

With QIAGEN Plasmid Kits, bacterial lysates are cleared by centrifugation. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and pure plasmid DNA is eluted in high-salt buffer (see flowchart "QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Applications

Plasmid DNA purified with QIAGEN Plasmid Kits is highly suitable for use in applications, such as:

This protocol is designed for the rapid, easy, and non-toxic preparation of up to 2 mg genomic DNA from not more than 2 g of tissue using QIAGEN-tip 2500. QIAGEN® Genomic-tips 20/G, 100/G, and 500/G can also be used with this protocol by reducing the amount of starting material according to the table on page 2. The purified genomic DNA ranges in size from 50-150 kb.

This procedure has been used successfully for isolation of 150-250 kb BAC DNA from a mouse-BAC library cloned in pBeloBAC11 from Escherichia coli strain HB101/r. The yield of BAC DNA from 100 ml culture was typically 20-40 μg.

The procedure has been used successfully for isolation of high- and low-copy-number plasmids from various Bacillus subtilis strains. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.

The procedure has been used successfully for isolation of 110 kb P1 DNA (pAdsacBII with an 80 kb insert) from Escherichia coli strain NS3529. Yield of P1 DNA was typically 10-50 µg from 500 ml culture.

The procedure has been used successfully for isolation of linear plasmids from Borrelia burgdorferi sensu lato species, which include Borrelia burgdorferi sensu stricto, Borrelia afzelli, and Borrelia garinii.

The procedure has been used successfully for isolation of cryptic plasmids (pLC2-based) from mesophilic Lactobacillus strains such as L. sake and L. curvatus. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.

The procedure has been used successfully for isolation of the large (128 kb), very-low-copynumber (1-2 copies per cell) plasmid pHCG3 and its derivatives from Oligotropha carboxidovorans. Yield of plasmid DNA was typically 3-6 µg plasmid DNA from 200 ml culture.

The procedure has been used successfully for isolation of a variety of medium-copy-number shuttle vectors from S. xylosus, S. carnosus, S. epidermidis, and S. aureus. Yield of plasmid DNA was typically 2-10 µg from 50 ml culture.

The procedure has been used successfully for isolation of different medium-copy-number plasmids carrying pHM1519 or pBL1 origins of replication from Corynebacterium glutamicum ATCC 13032. Yield of plasmid DNA was typically 0.4-1.5 µg per ml LB culture, although yield was dependent on the vector, the insert, and the size of the plasmid.